[CANCER RESEARCH 40, 288-292, February 1980 0008-5472/80/0040-0000$02.00 Correlation of the Production of Plasminogen Activator with Tumor Metastasis in Bi 6 Mouse Melanoma Cell Lines Bosco Shang Wang,2 Gerard A. McLoughlin,3 Jerome P. Richie, and John A. Mannick Deportment of Surgery, Peter Bent Brigham Hospital (B. S. W., G. A. M., J. P. R., J. A. M.j, and Department of Pathology (B. S. W.j, Harvard Medical School, Boston, Massachusetts 02115 ABSTRACT circulating cancer cells were easily detected in patients whose blood had a higher level of FA. The correlation between the production of plasminogen ac Although the FA of a tumor has been suggested as correlat tivator (PA) of tumors and their metastatic potential was stud ing with its metastatic potential, direct and quantitative evi ied. Bi 6 melanoma cells and ‘‘Bi6 mets' ‘cells (harvested from dence to support this hypothesis is lacking. Therefore, we have the pulmonary metastatic nodules of C57BL/6J mice bearing investigated this issue by comparing the FA of the Bi 6 mouse Bi 6 isografts) were examined with respect to their fibrinolytic melanoma and several of its sublines varying in their potential activity (FA) in tissue culture. Bi 6 mets cells had a significantly for forming metastases. higher FA than did Bi 6 cells. Fl (a Bi 6 subline with a lower incidence of metastasis) and Fl 0 (a highly metastatic Bi 6 subline) were also studied. Fl 0 cells produced more FA than MATERIALS AND METHODS did Fi cells. The difference between the FA's of these tumors Tumors. Bi 6 melanoma(The JacksonLaboratory,Bar Har was due to differences in their PA production. Significant bor, Maine) was maintained by serial transplantation either in differences in PA production between Fi and Fl 0 could be adult male C57BL/6J mice or in tissue cultures. ‘‘Bi6mets― consistently observed when i O@or more cells were cultured cell line was established in tissue culture by explantation of for at least 24 hr. The cell-free supernatants harvested from from 10 to 15 metastatic melanoma nodules from the lungs of 72-hr cultures of Fi 0 cells had a higher FA than those har C57BL/6J mice that had been inoculated s.c. with 1O@Bi6 vested from Fl cultures. Results suggest that a quantitative cells into the hind limbs 4 weeks previously. BI 6 mets subline difference in PA production between these 2 melanoma sub was further characterized as a melanoma by the presence of lines does exist and that it may contribute to their different melanin in the cytoplasm. The cell lines Fl and Fl 0 (kindly metastatic potential. supplied by Dr. I. J. Fidler, Frederick, Md.) were variants of the Bi 6 melanoma originally selected by explantation of succes INTRODUCTION sive generations of pulmonary metastatic cells, thus yielding 2 sublines with widely different metastatic potentials (6). Both Fl Most malignant tumors share a general pathogenesis of and Fl 0 cells have been maintained by weekly passages in blood-borne metastases. This process consists of local invasive tissue cultures with Roswell Park Memorial Institute Tissue growth of the primary tumor followed by vasculanization of the Culture Medium 1640 (Grand Island Biological Company, tumor mass and subsequent spread of tumor cells into the Grand Island, N. V.) supplemented with 10% heat-inactivated host's circulatory systems. Tumor cells that survive in the fetal calf serum, 2 mM L-glutamine (Grand Island Biological bloodstream may lodge at a distant site and develop into a Company), 100 units of penicillin per ml, and 100 @xgofstrep metastatic colony. Whether or not such a metastasis forms tomycin per ml over a period of 2 yr in our laboratory. depends upon a number of factors, among which the FA4 of Preparation of ‘[email protected] modified tech the tumor cells is considered to be important. nique of Moroz and Gilmore (16) was used. Two mg of 1251.. It seems logical, although still controversial, that tumors fibrinogen (specific activity, @l40Ci/mI; Abbott Laboratories, possessing a high FA should be able to digest surrounding North Chicago, III.) and 300 @gofpurified human plasminogen, fibrin and penetrate into the circulation much more easily than prepared by L-lysine Sepharose affinity chromatography (4), do tumors containing less FA. This theory is supported by a were dissolved in 100 ml of 0.01 M phosphate buffer (pH 7.4). study in which fibninolysis induced by prolonged treatment with To each 5-mI polystyrene tube, 0.2 ml of this mixture was heparin markedly increased metastatic spread in mice without added. Tubes were incubated at 37°with constant rotation for altering the primary tumor growth (22). A high level of FA has 3 hr. Tubes were washed 3 times with 0.05 M Tnis-O.1 M NaCI been observed in several malignant tumor cell lines (i 1—i3, (pH 7.4) buffer, and 6.25 mg of human serum albumin (Mas 20, 24) and also in normal embryo fibroblast cells, but only sachusetts Public Health Biological Laboratories, Boston, after these cells were transformed by oncogenic viruses (21, Mass.) in 0.5 ml of bufferwere added and incubatedat 25°for 23, 25). Clinically, Newstead et a!. (i 7). studied a group of 30 mm to occupy all unbound polystyrene sites. Tubes were patients with carcinoma of the colorectum and found that washed twice, and 5 units of thrombin (Topastasin; Roche Laboratories, Nutley, N. J.) in 0.1 ml were added and incubated I Supported by USPHS Grant CA21 644. at 37°for an additional 5 mm. Tubes were again washed 3 2 To whom requests for reprints should be addressed at the Peter Bent Brigham Hospital, 721 Huntington Avenue, Boston, Mass. 02115. times and were stored by filling with 4.5 ml of Tris-NaCI buffer 3 Recipient of Wellcome Travel Trust Grant. containing 0.1 % sodium azide at 4°.Thesetubes were washed 4 The abbreviations used are: FA, fibrinolytic activity; PA, plasminogen acti vator. 3 times with Dulbecco's phosphate-buffered saline (Grand Received March 16, 1979; accepted October 25, 1979. Island Biological Company) prior to use. The average cpm of 288 CANCERRESEARCHVOL.40 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1980 American Association for Cancer Research. PA Production and Tumor Metastasis newly prepared tubes were from 50,000 to 80,000, varying higher FA in vitro than did Bi 6 cells, even though both tumors from batch to batch. were harvested from the same animals and BI 6 mets were Fibrinolytic Activity of Tumor Cells In Vitro. Melanoma cells originally derived from Bl 6. were prepared either from C57BL/6J mice by the method of Comparison of Metastatic Potential and FA of Fl and FlO Hammond eta!. (10) or from tissue culture by trypsin treatment. Melanoma Sublines. In order to determinewhether or not the Cells were washed 3 times to remove trypsin completely and FA of Bi 6 sublines had a connection with their metastatic were then suspendedin complete Roswell Park Memorial In characteristics, 2 well-established melanoma sublines, Fl and stitute Tissue Culture Medium 1640 containing 10% fetal calf Fl 0, were used for subsequent studies. A group of mice were serum (from which plasmmnogenwas completely depleted by given injections of either Fl or Fl 0 cells into the left hind limbs. preheating to 56°for at least 45 mm twice), 2 mM L-glutamine, Ten days later, limbs were amputated after tumors became 100 units of penicillin per ml, and 100 @.sgofstreptomycin per palpable. Animals were killed at 6 weeks, and their lungs were ml. Cells were counted with a hemocytometer, and the viability removed and examined for evidence of metastases. As mdi was always greater than 98%, as was determined by trypan cated in Table 2, Fl had a low tendency of spontaneous blue dye exclusion. One ml of cell suspension with an appro pulmonary metastases as compared to Fl 0, which was highly priate number of tumor cells was added to each 125I-fibrin metastatic. This is in agreement with a previous observation by coated tube and was incubated at 37°in a 10% CO2 water Fidler (6). Furthermore, both tumor cells were obtained from saturated atmosphere for 24 hr. Tubes were centrifuged at culture flasks, and 1O@cellsin 1 ml of medium were immediately 800 x g for I 0 mm, and 0.2 ml of culture supernatant was added to each 125l-fibrin-coated tube. After incubation for 24 removed by a micropipet (Oxford Labs, Inc., Foster City, Calif.) hr, tubes were centrifuged, and 0.2 ml of supernatant was and counted in a gamma counter (Packard Instrument Co., removed and counted in a gamma counter. As shown in Table Downers Grove, Ill.). In some experiments, 10 x 1O@tumor 3, Fl 0 tumor cells had a significantly higher FA than did Fl cells were cultured in 20 ml of complete medium in a Falcon tumor cells in all 5 experiments performed (Experiments 1 to No. 3024 culture flask and were incubated for 72 hr. Cells 5). Experiments 6 to 9 were performed by incubating cell-free were removed by centrifugation, and the supernatant was supernatants harvested from 72-hr cell cultures of Fl or Fl 0. filtered through a 0.45-sm Millipore filter (Millipore Corp., Bed The FA of supernatants produced by Fl 0 was again signifi ford, Mass.). One ml of this cell-free supernatant was added to cantly greater than that of supernatants produced by Fl in 3 of each 125l-flbrmn-coatedtube,tubes were incubated for an ad 4 experiments.